Method of culturing human chondrocytes

ABSTRACT

This invention provides a method for rapidly culturing a large amount of human chondrocytes to give normal chondrocytes or a mass thereof. The culture method comprises co-culturing human chondrocytes together with perichondral cells in the chondrogenic stage, as feeder cells, which support the proliferation ability of the chondrocytes, to allow rapid culturing of the human chondrocytes in a large amount.

TECHNICAL FIELD

[0001] This invention relates to a novel method of culturing normalhuman chondrocytes and to the normal human chondrocytes obtained by themethod. This invention also relates to a cartilage therapy materialusing the normal human chondrocytes obtained by the method.

BACKGROUND ART

[0002] In living body, chondrocytes exist integrated in the matrix ofthe living cartilage and can be isolated from the matrix by treating thetissues with an enzyme such as collagenase. One of the methods oftherapy devised in the field of treating cartilage-related diseases hasbeen transplanting of isolated chondrocytes, especiallyautotransplantation of chondrocytes. For this purpose, trials conductedin animal experiments have attempted transplanting of isolatedchondrocytes into defective articular cartilage (Bently, et. al., Nature230, 385-388 (1971); Langer, Clin. Orthop. 124, 237-250 (1977); Astonet. al., J. Bone Joint Surg. 68-B, 29-35 (1986); Wakitani, et. al., J.Bone Joint Surg. 71-B, 74-80 (1989)).

[0003] Normal chondrocytes are required in large amounts forautotransplantation, but since only a small amount of cartilage can beextracted in the case of humans, it has been very difficult to obtainchondrocytes in a sufficient amount required for transplant. It has beenattempted in the past to culture human chondrocytes of articularcartilage, auricular cartilage and costal cartilage. However, due to theminimal amount of human chondrocytes that can be used for culturing andthe lack of an effective established method for culturing humanchondrocytes, it has either been impossible to maintain the primaryculture, or else when the primary culture has been successful, the timerequired has been very long, from six to eight weeks (Brittberg, et.al., New Engl. J. Med. 331, 889-895 (1994); Aulthouse, et. al. In vitroCell. & Develop. Biol. 25, 659-668 (1989); Ting, et. al., 40, 413-421(1998); Rodriguez, et. al., Plast. Reconstr. Surg. 103; 1111-1119(1999)).

[0004] On the other hand, in cases of animals other than human (mouse,rabbit, chicken, cow, etc.) culturing of chondrocytes has beensuccessful in some cases because large amounts of chondrocytes areavailable for primary culture and chondrocytes are easily extracted fromfetuses or the young, which have extremely soft cartilage (Bently et.al. Nature 230, 385-388 (1971); Langer, Clin. Orthop. 124, 237-250(1977); Aston et. al., J. Bone Joint Surg. 68-B, 29-35 (1986); Wakitani,et. al., J. Bone Joint Surg. 71-B, 74-80 (1989)).

DISCLOSURE OF THE INVENTION

[0005] This invention therefore aims to provide a method of rapidlyculturing human chondrocytes to obtain a large amount of normalchondrocytes and a mass thereof. This invention also aims to providecartilage therapy materials using the obtained normal human chondrocytesor a cell mass thereof.

[0006] The inventor of this invention, as a result of intensiveresearch, discovered that perichondral cells in the chondrogenic stagesupport the proliferation ability of human chondrocytes. It was alsodiscovered that co-cultivation of human chondrocytes together with suchperichondral cells used as feeder cells can rapidly produce a largeamount of human chondrocytes, especially in primary culture. It wasfurther discovered that normal characteristics are maintained in thechondrocyte on mass obtained by multilayer seeding and further culturingof the thus cultured human chondrocytes.

[0007] This invention therefore provides a method of rapidly culturing alarge amount of human chondrocytes by co-culturing between humanchondrocytes and perichondral cells in the chondrogenic stage, which areused as feeder cells to support the proliferation of human chondrocytes.Furthermore, a chondrocyte mass in a gel state can be obtained by multiseeding and culturing of the thus obtained human chondrocytes one ormore times. The number of times of multilayer seeding will depend on thesize of cultured cartilage tissue required, but from 3 to 4 times isgenerally preferable.

[0008] It is preferable to use chondrogenic-stage perichondral cellsfrom a mammalian fetus as feeder cells for the human chondrocyteculturing method of this invention, and it is more preferable to usefirst arch cartilage, Meckel's cartilage perichondral cells. Especiallypreferable are chondrogenic-stage perichondral cells from a 13-day-oldmurine fetus.

[0009] The mammalian fetus used for the invention can be a fetus of anymammal, for example, a rodent (mouse, rat, rabbit, etc.), dog, ape, cow,goat or sheep. It is preferable to use a mammal such as mouse, for whichdetailed biological research on the viviparous period has beenconducted.

[0010] This invention also provides normal human chondrocytes obtainedby the method of invention, and a cell mass thereof. The humanchondrocytes obtained by the method of the invention are integrated intothe cartilage matrix which contains chondrocalcin, type II collagen andthe like. The cultured chondrocytes also bind to each other through thecartilage matrix to form a gel-like cell mass. For the purpose of thisinvention, “normal human chondrocytes” means chondrocytes that maintainthe characteristics of the original cartilage.

[0011] This invention also provides cartilage therapy materialsconsisting of materials incorporating the above-mentioned humanchondrocytes or their cell mass. Any artificial or natural material canbe used as the material incorporating the human chondrocytes or cellmass so long as it protects the chondrocytes for transplant from thesurroundings and prevents them from diffusing and being absorbed. Thosewhich can serve as scaffolding for the chondrocytes to be transplantedare preferred. Examples of such materials are collagen, polyglycolicacid (PGA), polylactic acid, alginates (for example, the calcium salt),polyethylene oxide, fibrin adhesive, polylactic acid-polyglycolic acidcopolymer, proteoglycans, glycosaminoglycans and human dermis. Aproteoglycan or glycosaminoglycan may be sulfated.

[0012] This invention also includes treatment methods forcartilage-related diseases, that employ the cartilage therapy materials.

[0013] This invention also relates to the use of perichondral cells inthe chondrogenic stage, which support the proliferation ability ofchondrocytes, as feeder cells in the above-mentioned treatment method.

[0014] Human Chondrocytes

[0015] The method of the invention can be applied to the cultivation ofhuman chondrocytes of any cartilage tissue such as auricular cartilage,costal cartilage, articular cartilage, intervertebral cartilage, ortracheal cartilage. Chondrocytes of auricular cartilage, costalcartilage and articular cartilage are especially suitable. Any articularcartilage, from the mandibular joint, upper arm joint, elbow joint,shoulder joint, hand joint, hip joint, waist joint or leg joint, can beused.

[0016] The chondrocytes used in the cultivation method of the inventioncan be isolated from human cartilage tissue by conventional methods.Chondrocytes are usually isolated by chopping the sampled cartilagetissue with a chopping knife or the like, treating the chopped cartilagewith trypsin or collagenase (e.g. type II collagenase) and filtering toisolate the chondrocytes. The preferred procedure is as follows.

[0017] (1) The sampled cartilage tissue is disinfected with anantibiotic (for example, penicillin, kanamycin) or an antifungal agent(for example, amphotericin B) and then minced with a scalpel or achopping knife.

[0018] (2) The minced cartilage tissue is treated with EDTA, keptstationary overnight in a medium containing trypsin (preferably at 4°C.), transferred into a medium containing type II collagenase (and keptstationary for 1 to 6 hours, if necessary) and incubated for 1 to 6hours at 37° C.

[0019] (3) The treated cartilage is transferred to a culture mediumcontaining fetal bovine serum and agitated gently in the culture mediumfor several hours. The resulting culture medium is filtered with afilter (for example, a 100-micrometer filter) to isolate thechondrocytes.

[0020] By this method, 0.5-1.0×10⁶ human chondrocytes can be obtainedfrom 0.5 cm² of human cartilage tissue. The cultivation method of thisinvention can also be combined with known growth factors, especiallythose which stimulate proliferation of cartilage, such as FGF (forexample, bFGF), IGF (for example, IGF-I) and/or bone morphogeneticprotein 9 (BMP9).

[0021] Feeder Cells

[0022] Feeder cells are cells that contribute to cell cultivation bysupporting the proliferation ability and differentiation ability of aspecific tissue or cell. For example, 3T3 cells derived from fibroblastcells are known as a feeder cells which support the proliferationability of epidermal keratinocytes (Green et. al., Cell 6, p.331-344(1975)). However no feeder cells have been known for chondrocytes, andespecially for human chondrocytes.

[0023] The feeder cells used for the cultivation method of thisinvention are cells which support the proliferation ability of humanchondrocytes when they are co-cultured and which allow the proliferatedchondrocytes to maintain the characteristics of the original cartilagetissue, or specifically, to maintain the characteristics of sampledcartilage tissues.

[0024] Such feeder cells can be obtained by screening cells using theabove-mentioned criteria, i.e. supporting of the proliferation abilityof human chondrocytes, and maintenance of characteristics. In a concretescreening procedure, sample cells are co-cultured with humanchondrocytes, it is examined whether the chondrocytes have proliferatedand whether the extracellular matrix of the proliferated cells is thesame as the original cartilage matrix. Cells to be screened can be thoseisolated from a living body (including a fetus or embryo) or a tissuethereof, or a cloned cell. Chondrogenic-stage perichondral cells from ananimal, especially a mammal, will be referred to as the exemplary feedercells of the invention.

[0025] It is preferable to use chondrogenic-stage perichondral cellsfrom a mammal for which detailed biological research on the viviparousperiod has been conducted, e.g. a rodent (mouse, rat, rabbit), andespecially chondrogenic-stage perichondral cells from a mouse. Apreferred chondrogenic stage is the period of differentiation andformation of first arch cartilage-Meckel cartilage. For a mouse, thiscorresponds to a 13-day-old fetus. It is also possible for thisinvention to use first arch cartilage-Meckel perichondral cells of avertebrate other than a mammal, for example, the embryo of a bird(chicken, etc.) as the feeder cells.

[0026] Although the feeder cells can be used for seeding without anytreatment after they are isolated from the fetus or embryo, it ispreferable to culture them in a suitable culture medium for about 10 to14 days before use. The feeder cells can be subcultured (up to a 5^(th),and preferably 1^(st)-3^(rd) subculture), and preserved in a frozenstate, preferably at a temperature and under conditions where the cellorganelles are not destroyed. An example of such preservation is storagein DME medium containing a cryo-protectant such as 10% FBS, 10%glycerin, at −130° C. or below. When the feeder cells are subcultured,it is suitable to change the medium about 3 times a week. Thecryopreserved cells are thawed for use as the feeder cells.

[0027] Even though the feeder cells are used in the primary culturetogether with the chondrocytes, in some cases the proliferation ofchondrocytes may be weak, in which case co-culturing may only bepossible up to the 1^(st) subculture. It is therefore most effective andpreferable to use the feeder cells at the time of primary culture.Before the co-culturing with chondrocytes, it is possible to eliminatethe proliferation ability of the feeder cells by radiation (e.g. gammarays, cobalt radiation) or chemical treatment. The chemical treatmentcan be performed with an anti-cancer agent such as mitomycin C oractinomycin D, or an antibiotic such as ampicillin.

[0028] Method of Culturing Human Chondrocytes

[0029] Conventional culture media suitable for cultivation ofchondrocytes can be used for co-culturing the human chondrocytes andfeeder cells (mainly in primary culture). In addition to hydrocortisone(HC) and fetal bovine serum (FBS) into the media, a proliferation factorsuch as human bFGF or human IGF-I is added (Cuevas et. al., Biochem.Biophys. Res. Commun. 156, 611-618 (1988); Froger-Gaillard et al.,Endocrinology, 124, 2365-2372). An example of such a medium is a mixedmedium of F-12 and DME (H) in a ratio of 1:1, with addition of FBS(preferably about 10%), human bFGF (preferably about 10 ng/ml), HC(preferably about 40 ng/ml) and human IGF-I (preferably about 5 ng/ml).

[0030] 1) Primary Culture

[0031] The feeder cells pre-processed by radiation are seeded in a flask(in the case of a flask having a 75 cm² base area, a concentration of1.0×10⁶ cells is preferable), and the chondrocytes are seeded(preferably 0.5-1.0×10⁶ cells) and then cultured at conditions suitablefor the cultivation of the chondrocytes (e.g. 37° C., 10% CO₂). Thecultivation is continued until the proliferated cells form a confluentmonolayer (usually for 10-14 days).

[0032] 2) Subculture

[0033] The subculture can be performed with the same culture medium asthe primary culture (usually seven days for one subculture). When thecells obtained by primary culture are subcultured, in the case ofauricular cartilage, the number of cells increases by 30 to 746 timesfrom P0 (primary culture)→P6. In the case of costal cartilage, thenumber of cells increases by 65 to 161 times from P0→P4. When morechondrocytes are desired, the number of subcultures can be increased.

[0034] 3) Multilayer Culturing

[0035] A gel-like chondrocyte mass can be obtained by multilayer seedingand culturing of chondrocytes obtained by one or more seedings, andpreferably 3 to 4 seedings. In the obtained chondrocyte mass, the humanchondrocytes are surrounded and connected by a cartilage matrixcontaining chondrocalcin and type II collagen to form a gel-like cellmass. As mentioned above, the chondrocytes obtained by the cultivationmethod of this invention maintain the characteristics of the originalcartilage tissue, i.e. the cartilage tissue used for the primaryculture. Maintenance of characteristics of the original cartilage tissuecan be confirmed by the morphology of the cultivated cells and thecomposition of extracellular matrix (for example, chondrocalcin, type IIcollagen, as well as hyaluronic acid, chondroitin, chondroitin sulfateand keratosulfate).

[0036] Cartilage Therapy Material

[0037] The human chondrocytes or the cell mass obtained by the inventionis integrated into a biomaterial and used for transplant as a cartilagetherapy material. Examples of biomaterials into which human chondrocytesor their cell mass may be integrated include: collagen, polyglycolicacid (PGA), polylactic acid, alginates (for example, the calcium salt),polyethylene oxide, fibrin adhesive, polylactic acid-polyglycolic acidcopolymer, proteoglycans, glycosaminoglycans and human dermis, which maybe used independently or in combination. Proteoglycans andglycosaminoglycans maybe sulfated. Those biomaterials separate thechondrocytes from the surroundings and prevent the chondrocytes fortransplant from diffusing or being absorbed, and preferably can serve asa scaffold for the transplanted chondrocytes. The biomaterials can be inany form, for example, membrane such as sheet, porous bodies such assponges, meshes such as knits, textiles, non-woven fabrics, cotton andthe like. It is preferable to use a porous material because this willallow the chondrocytes to more easily adhere to the biomaterial andpermeate inside to promote the formation of cartilage tissue.

[0038] When collagen is used as a biomaterial, it can be cross-linked.With increased strength by the cross-linking, the cartilage therapymaterial composed of the collagen can be stably fixed to the body by astaple or the like and can support the chondrocytes or cell mass withthe necessary strength until the new cartilage tissue forms.

[0039] Moreover, the collagen can be solubilized to form a collagensolution for admixture with the chondrocytes or cell mass, and themixture gelled, if necessary, for use as a cartilage therapy material.Conventional collagen solutions can be used and it is preferable to usean enzyme-solubilized collagen solution because this removes immunogenictelopeptides. It is also possible to form a cartilage therapy materialby integrating or overlaying a mixture of collagen solution andchondrocytes into or onto a sponge, non-woven fabric or the-like.

[0040] When the materials into which the chondrocytes are integrated areproperly selected, combined and shaped into a supporting body, thecartilage therapy material can not only form cartilage tissue but canalso induce cartilaginous ossification. Human dermis is an example ofsuch a material which induces cartilaginous ossification. Ossificationcan also be promoted by a growth factor which promotes bone formation,such as bone morphogenetic protein (BMP).

[0041] The invention will now be illustrated in detail by Examples.However, the invention is not restricted to these Examples.

BEST MODE FOR CARRYING OUT THE INVENTION EXAMPLES Example 1

[0042] Chondrocyte Cultivation

[0043] Culture-medium composition: To a mixed medium of F-12 and DME (H)in a ratio of 1:1 there were added 10% FBS, 10 ng/ml human bFGF (KakenPharmaceutical Research Institute), 40 ng/ml HC and 5 ng/ml human IGF-I(Gibco).

[0044] Chondrocytes: Chondrocytes were isolated from rear auricularcartilage (about 1×1 cm) and from precordial costal cartilage (about 1×1cm) respectively.

[0045] Feeder cells: Cells surrounding first arch cartilage-Meckelcartilage of a 13-day-old mouse fetus were collected and cultivated toproduce feeder cells, designated as CS21 cells.

[0046] (a) Isolation of Chondrocytes

[0047] A piece of cartilage was disinfected with Penicillin G (100 u/ml)and kanamycin (0.1 mg/ml) and processed with 0.02% EDTA for 15 minutes.The piece of cartilage was then chopped with a scalpel and choppingknife, kept stationary overnight at 4° C. in a medium containing 0.25%trypsin, and then incubated at 37° C. for 1-6 hours in DME mediumcontaining 0.3% type II collagenase. It was then transferred into DMEmedium containing 10% FCS, gently agitated with a stirrer for 2 hoursand filtered with a 100-micrometer filter to separate the chondrocytes.

[0048] (b) Feeder Cells

[0049] i) Isolation of Feeder Cells

[0050] Cells surrounding first arch cartilage (Meckel cartilage) of a13-day-old (chondrogenic stage) live Balb mouse fetus were collectedusing microsurgery technology. The obtained cell mass was transferred toa calcium-free culture medium containing 0.125% trypsin, left stationaryin a refrigerator at 4° C. for 4 hours, agitated gently by a stirrer for10 minutes and filtered to isolate single cells.

[0051] ii) Culture Conditions

[0052] The feeder cells were cultured in DME medium containing 5-10% FBS(fetal bovine serum). In primary culture, they became confluent in 3-4days and in the first subculture they became confluent in 3-5 days. Fromthe second subculture, the cells were subcultured every week and the1^(st) to 3^(rd) subcultures were cryopreserved. The obtained cells (CS21 cells) were used as feeder cells.

[0053] (c) Primary Culture

[0054] CS21 cells treated with 60 Gy of radiation (cobalt) on theprevious day were seeded on a flask having a base area of 75 cm² at adensity of 1.0×10⁶ cells/cm². On the same flask, the isolated humanchondrocytes were also seeded at a density of 0.5-1×10⁶ cells/cm², andcultured at 37° C. under 10% CO₂. As a control, 3T3 cells were used asfeeder cells under the same conditions. In the primary culture, theculture medium was exchanged two times a week. As a result, thechondrocytes co-cultured with CS21 cells became confluent in monolayerupon culturing for 10-14 days, whereas little growth was observed when3T3 was used (FIG. 1). The obtained cells were used for the followingsubculture.

[0055] (d) Subculture of Chondrocytes

[0056] Subculture was carried out under the same conditions as theprimary culture except that feeder cells were not used and the period ofone subculture was 7 days. The human chondrocytes obtained by theprimary culture were seeded on a flask at a density of 1.0×10⁶ cells/175cm² of the base area, subcultured every 7 days and cryopreserved aftereach subculture, and the increased number of chondrocytes was counted.

[0057] In the case of auricular cartilage (n=20), the cultivation wascarried out by primary culture of auricular cartilage cells (n=20) P0→P1(n=20) P1→P2 (n=13) P2→P3 (n=12) P3→P4 (n=9) P4→P5 (n=6) P5→P6 (n=3),and cells obtained at each subculture were cryopreserved. The cell countincrease was about 30- to 746-fold.

[0058] In the case of the costal cartilage (n=12), the cultivation wascarried out by the primary culture of costal cartilage cells (n=12)P0→P1 (n=12) P1→P2 (n=6) P2→P3 (n=4) P3→P4 (n=2), and the cells werecryopreserved in a similar manner. The cell count increase was about 65-to 161-fold.

[0059] When the concentrations of chondrocalcin, which is a component ofcartilage matrix, in the culture media were measured by enzymeimmunoassay (EIA) using anti-chondrocalcin antibody, chondrocalcin wasdetected at a concentration higher than 100 ng/ml both with theauricular cartilage and costal cartilage which were subcultured for 7days. On the other hand, almost no cell proliferation was observed inthe case of each chondrocytes cultured with no feeder cells or 3T inprimary culture. The results of the cell cultures are shown in thefollowing table 1. TABLE 1 Cartilage tissue Auricular cartilage Costalcartilage Feeder cell CS21 CS21 cells Not used cells Not used CellPrimary ++ − ++ − proliferation culture Subculture +++ NT* +++ NT*

[0060] The results show that primary culture by the method of theinvention can be achieved in 2 weeks, the cell proliferation rate ishigh, and a large amount of normal cells are rapidly cultured, whileconventional culture-system without feeder cells cannot be maintainedbecause establishing primary culture is difficult, and even ifsuccessful, it takes 6-8 weeks for primary cultivation. Furthermore, thefact that a large amount of chondrocalcin of cartilage matrix waspresent in the culture media suggests that the increased cells arechondrocytes.

[0061] (e) Multilayer Culturing

[0062] The chondrocytes obtained by the subculture were seeded byoverlaying 3 times at a density of 1×10⁶ cells/cm² and cultured bymultilayer culturing. A sheet-like gel mass was formed after two weeksof culturing (FIG. 2). When the gel-like chondrocyte mass was stained byHE (hematoxylin-eosine) staining, the cells were seen to be multilayeredand joined together through the matrix (FIG. 3). Moreover, whenimmunological staining was performed for type II collagen, which is amolecular marker of cartilage tissue, the extracellular matrix exhibitedstaining indicating that the extracellular matrix was acartilage-specific matrix (FIG. 4).

Example 2

[0063] Transplanting of Chondrocytes

[0064] (a) In order to examine whether cartilage is formed without theinfluence of chondrocyte-integrating material (support) the obtainedhuman chondrocyte mass was meshed in glass fiber (filter) andtransplanted under the dorsal skin of nude mice (BALB/C Sic/nu, 10-weekold, 25 g) (n=10). Formation of cartilage tissue was confirmed in 3/10mice. Since large tissue did not form, it was conjectured that thechondrocytes were not stably held by the glass fiber and diffused intothe surrounding tissues. Moreover, when the chondrocyte mass alone wastransplanted, the chondrocytes were found diffused under the skin. Itwas thus shown that the chondrocyte mass for transplant must beprotected from the surroundings.

[0065] (b) The human chondrocytes were held in PGA (n=10) as a support,in such a manner that the chondrocytes were incorporated in the support,and transplanted into nude mice in the same manner. As a result,formation of cartilage tissue was observed in 100 percent of the samples(10/10). FIG. 5 shows the chondrocyte masses used for transplant, FIG. 6shows a mouse three months after the transplant and FIG. 7 shows thespecimens of the transplanted tissue.

[0066] Moreover, when calcium alginate (n=5) was used as a support,formation of cartilage tissue was also observed in 100 percent of thesamples (5/5). The specimens using PGA and calcium alginate as supportwere then made into preparations for microscopy and stained withToluidine Blue. As a result, the Toluidine Blue metachromacia specificto cartilage tissue was observed (FIG. 8). Since larger cartilage tissuewas formed with PGA, it is conjectured that PGA is more suitable as asupport.

[0067] (c) The conditions for ossification of the formed cartilagetissue were examined next. (Group 1) A human chondrocyte mass wasintegrated into PGA and covered with fibrin. (Group 2) Another humanchondrocyte mass was integrated into PGA, wrapped with human dermis andthen covered with fibrin. Both groups were transplanted under the dorsalskin of nude rats (F344/N rnu/rnu, 6-week-old, 250 g) and formation ofcartilage tissue was examined in both groups. Specimens extracted 3months after the transplant showed Toluidine blue metachromacia specificto cartilage tissue, thus confirming that cartilage tissues had formedin both groups (FIG. 9). In Group 1, in which chondrocyte mass wasintegrated with PGA alone, only cartilage tissue formation wasconfirmed. In Group 2, however, in which the mass was wrapped withdermis, vascularization was also observed in the perichondrium andmatrix, and cartilaginous ossification was further observed in parts ofthe cartilage (FIG. 10).

[0068] (d) It was then examined whether the cartilage tissue formedafter the transplant expressed the characteristics of its origin (thecartilage tissue used for cultivation). Preparations of theabove-mentioned specimens were stained with HE (hematoxylin-eosine)staining, Toluidine Blue staining, Alcian blue+PAS staining and EVG(Elastica von Gieson) staining. The cell membrane and interstitium arestained by HE staining, thus allowing observation of cell morphology andconfirmation of cartilage tissue formation from the cartilage cavity.The metachromatism observed by Toluidine Blue is specific to cartilage.Alcian blue+PAS staining dyes polysaccharides, thus allowingconfirmation of a high amount of cartilage matrix, and especiallyelastin (glyoprotein) of elastic cartilage. EVG staining dyes elasticfiber, thus allowing confirmation of elastic cartilage (auricularcartilage) formation.

[0069] In the case of the cartilage tissue formed from the culturedauricular chondrocytes transplant, the elastic fiber was stronglystained by the EVG staining, and PAS staining was positive in alcianblue+PAS staining, thus identifying them as auricular cartilage cells.In the case of the tissue formed from the cultured costal cartilagetransplant, the elastic fiber was weakly stained by EVG staining, whilePAS staining was positive in alcian blue+PAS staining, therebyidentifying them as costal cartilage cells.

[0070] (e) When a similar transplant experiment was carried out usingcollagen sponge instead of PGA as support, formation of cartilage tissuewas observed in 100 percent of the samples of auricular cartilage (5/5).The transplant specimens were made into preparations and stained with HEstaining or alcian blue+PAS staining, and it was confirmed thatcartilage tissue had formed (positive in HE staining) and that theformed cartilage tissue was auricular cartilage (positive in alcianblue+PAS staining) (FIG. 11). Formation of cartilage tissue was alsoconfirmed when the same experiment was repeated with costal cortilage.

[0071] (f) To confirm that the tissues after the transplant were humanderived, antibody staining was carried out by the ABC-PC (peroxidase)method using anti-human type II collagen antibody. As a result,expression of human type II collagen was observed in both auricularcartilage and costal cartilage, thus confirming that the tissues were ofhuman origin (FIG. 12). The results are shown in the following table.TABLE 2 Auricular Costal cartilage cartilage HE staining + + ToluidineBlue + + metachromatism PAS staining ++ + EVG staining ++ +/− Human typeII collagen + +

[0072] (g) To further confirm that the formed cartilage tissue was ofhuman origin, RT-PCR was performed with type II collagen. RT-PCR wascarried out using the following primers having nucleotide sequencesspecific to human or mouse type II collagen, and the PCR products weretreated with the restriction enzyme EcoRI. Human; Forward primer:5′-ACATACCGGTAAGTGGGGCAAGAC-3′ (SEQ ID NO: 1) Reverse primer:5′-AGGTCTTACAGGAAGACAATAAAT-3′ (SEQ ID NO: 2) Mouse; Forward primer:5′-ATTTTGCAGTCTGCCCAGTTCAGG-3′ (SEQ ID NO: 3) and Reverse primer:5′-AGGTCTTACAGGAAGACAATAAAT-3′ (SEQ ID NO 4)

[0073] Since human-derived type II collagen is cleaved by EcoRI, it canbe easily identified whether it is of human origin or mouse origin.RT-PCR was performed using samples 3 months after transplant from micetransplanted with cultured human chondrocytes tissue, with feeder cellsalone and with feeder cells+cultivated human chondrocytes, and, as acontrol sample, mouse cells. A conventional protocol was used forextracting the RNA from the samples and for the RT-PCR, according to“Gene Manipulation Manual” printed by Igaku-Shoin (published in 1995).

[0074] With the sample transplanted of cultured human chondrocytetissue, two bands were observed, indicating only human type II collagen.With the samples transplanted with feeder cells alone, digestion byEcoRI did not occur, indicating only mouse type II collagen. With thesample transplanted with the feeder cells+cultivated human chondrocytes,a band for cleavage by the restriction enzyme and a band for no cleavagewere observed, indicating both mouse and human type II collagen. Withmouse cells as the control, digestion by EcoRI did not occur, indicatingonly mouse type II collagen. These results confirmed that the cartilagetissue formed after transplant was of human origin. The results areshown in FIG. 13.

INDUSTRIAL APPLICABILITY

[0075] The present invention enables rapid culturing of large amounts ofhuman chondrocytes by co-culturing human chondrocytes with perichondralcells in the chondrogenic stage, which are employed as feeder cells tosupport the proliferation ability of the chondrocytes. A humanchondrocyte mass can be obtained by multilayer seeding and culturing ofthe obtained human chondrocytes one or more times. Cartilage therapymaterials in which human chondrocytes or their cell mass is integratedcan be produced. Appropriate selection of the material in which thechondrocytes are integrated allows the cartilage therapy material toinduce not only generation of cartilage tissue, but also cartilaginousossification. They are therefore useful for the treatment of diseasesinvolving cartilage and bone.

BRIEF DESCRIPTION OF THE DRAWINGS

[0076]FIG. 1 is a set of photographs showing the results of the primarycultures of human chondrocytes together with (A) feeder cells (CS21cells) of the invention and (B) 3T3 cells.

[0077]FIG. 2 is a photograph showing culture medium alone (left) and theresult of multilayer cultivation (right).

[0078]FIG. 3 is a microphotograph showing HE (hematoxylin-eosine)staining of a gel-like cell mass obtained by multilayer cultivation.

[0079]FIG. 4 is a microphotograph showing immunological staining usinganti-type II collagen of a gel-like cell mass obtained by multilayercultivation.

[0080]FIG. 5 is a set of photographs of (A) a material consisting of PGAas a support on which a human chondrocyte mass was overlaid and (B) amaterial consisting of PGA in which a chondrocyte mass was held.

[0081]FIG. 6 is a photograph of a mouse three months after a chondrocytemass was transplanted.

[0082]FIG. 7 is a set of photographs showing specimens extracted fromthe mice three months after transplant of the chondrocyte masses.

[0083]FIG. 8 is a set of microphotographs of extracted specimens stainedwith Toluidine Blue, wherein the supports were PGA (A) and calciumalginate (B).

[0084]FIG. 9 is a photograph of extracted specimens of a group in whicha chondrocyte cell mass was integrated in PGA (Group 1) and a group inwhich it was further wrapped with human dermis (Group 2).

[0085]FIG. 10 is a microphotograph showing the tissue of Group 2 inwhich human dermis was used for wrapping.

[0086]FIG. 11 is a set of microphotographs of (A) HE staining and (B, C)alcianblue+PAS staining of auricular cartilage tissue transplanted withcollagen sponge as a support in which chondrocyte mass was integrated.

[0087]FIG. 12 is a microphotograph of immunological staining oftransplanted auricular cartilage tissue using anti-type II collagen.

[0088]FIG. 13 is a photograph showing the result of RT-PCR for type IIcollagen of transplanted cartilage tissue.

1 4 1 24 DNA Artificial Sequence PCR primer 1 acataccggt aagtggggca agac24 2 24 DNA Artificial Sequence PCR primer 2 aggtcttaca ggaagacaat aaat24 3 24 DNA Artificial Sequence PCR primer 3 attttgcagt ctgcccagtt cagg24 4 24 DNA Artificial Sequence PCR primer 4 aggtcttaca ggaagacaat aaat24

1. A method for culturing human chondrocytes characterized byco-culturing human chondrocytes together with perichondral cells in thechondrogenic stage, as feeder cells, which support the proliferationability of chondrocytes, to allow rapid culturing of the humanchondrocytes in a large amount.
 2. The method of claim 1 characterizedin that the feeder cells are chondrogenic-stage perichondral cells of amammalian fetus.
 3. The method of claim 2 characterized in that theperichondral cells are first arch cartilage/Meckel perichondral cells.4. The method of claim 2 characterized in that the perichondral cellsare perichondral cells of a 13-day-old mouse live fetus.
 5. The methodof any one of claims 1-4 characterized by multilayer seeding andculturing of the cultured cells one or more times to obtain a gel-likechondrocyte mass.
 6. Normal human chondrocytes produced by the methoddescribed in any one of claims 1-5.
 7. Normal human chondrocytesproduced by the method described in any one of claims 1-5 and wrapped ina cartilage matrix comprising chondrocalcin and/or type II collagen. 8.Normal human chondrocytes of claim 6 or 7 which bind together throughthe matrix and form a gel-like cell mass.
 9. The cartilage therapymaterial consisting of material in which chondrocytes described in anyone of claims 6-8 are integrated.
 10. The cartilage therapy material ofclaim 9 wherein the material is collagen, polyglycolic acid, polylacticacid, an alginate, polyethylene oxide, fibrin adhesive, polylacticacid-polyglycolic acid copolymer, a proteoglycan, a glycosaminoglycanand/or human dermis.
 11. Feeder cells supporting the proliferationability of chondrocytes for rapid culturing of human chondrocytes in alarge amount by co-culturing with human chondrocytes.
 12. The feedercells of claim 11 which are chondrogenic-stage perichondral cells of amammalian fetus.
 13. The feeder cells of claim 12 wherein theperichondral cells are first arch cartilage/Meckel perichondral cells.14. The feeder cells of claim 12 wherein the perichondral cells areperichondral cells of a 13-day-old mouse live fetus.